Cell and Tissue Journal

Abstract Introduction: Mesenchymal stem cells (MSCs) are  important cellular components in the microenvironment of various types of cancers, including multiple myeloma (MM); their dynamic interactions with malignant cells can control the tumor microenvironment (TME) to favor the progression, drug resistance, and survival of cancer cells. It is shown that not only are MM cells influenced by the environmental cells, but also malignant cells affect the behavior of other cells in the TME. Secreted signaling factors are important players in this scenario. Accordingly, MSCs derived from TME, affected by cancer cells, are different from normal MSCs; therefore, in cases of disease modeling and drug screening studies, optimization of MSCs culture condition and their priming with cancer cell-derived secretome are crucial considerations to become more similar to the patient-derived MSCs. In this regard, conditioned medium, which is derived from the supernatant culture medium of cultured cells, contains cell-secreted factors and can be used as a source of cell secretome.
Aims: This study attempted to evaluate the effect of conditioned medium derived from the myeloma cell line U266 (U266-CM) on the proliferation, cell cycle, and differentiation of bone marrow-derived MSCs (BMMSCs).
Materials and methods: U266 cells were cultured in RPMI-1640 complete culture medium, and their conditioned medium was collected after two days of culture and stored. Since the basal culture medium of the U266 cell line (RPMI-1640) was different from the basal culture medium of BMMSCs (DMEM), in the first step, the impact of changing the culture medium from DMEM to RPMI-1640 on the proliferation and viability of BMMSCs was evaluated. In the next step, the effects of U266-CM treatment on the proliferation, cell cycle, and differentiation of BMMSCs into osteoblasts and adipocytes were assessed. Alkaline phosphatase activity assessment and Alizarin red staining were performed to evaluate the osteogenic differentiation, and Oil red O staining was carried out to assess the adipogenic differentiation. BMMSCs cultured in DMEM complete media were considered as control group.
Results: Changing the culture media from DMEM complete media to RPMI-1640 complete media affected the viability and proliferation rate of BMMSCs; however, up to 48 hours, the viability and proliferation rate of cells cultured in both culture media were maintained. Therefore, 48 hours was selected as the optimum incubation time of BMMSCs with U266-CM. Treatment with U266-CM decreased the proliferation rate of BMMSCs, assessed by cell counting and cell cycle analysis. Moreover, BMMSCs cultured in U266-CM showed slightly increased osteogenic differentiation and maintained their adipogenic propensity.
Discussion: Our results showed that CM derived from the MM cell line affected the proliferation rate and the differentiation potential of BMMSCs. These observations highlighted the importance of a more similar recapitulation of BMMSCs culture condition to the TME, for disease modeling and drug screening studies. Further experiments, including providing dynamic interactions of MSCs with MM cells in direct or indirect co-culture systems, are required to better recapitulate TME conditions for MSC priming. Moreover, for mechanistic analysis, evaluation of the secreted factors and assessment of expressed transcripts and proteins are highly recommended for future studies.
Conclusion: In summary, U266-CM influenced the proliferation rate and the differentiation potential of BMMSCs.

Abstract Aim: Hairy roots induced by inoculation with Agrobacterium rhizogenes are suitable for the production and accumulation of plant secondary metabolites. Genetically modified hairy roots produce valuable secondary metabolites to a greater amount and with more genetic stability than cell suspension cultures and normal roots. Catharanthus roseus is one of the most important medicinal plants that produces valuable secondary metabolites such as indole alkaloids. The purpose of this research was to investigate the effect of factors influencing the induction of hairy roots and to investigate the amount of production of secondary metabolites (vindoline and catharanthine) in the hairy roots produced by inoculation of C. roseus plant with A. rhizogenes.
Material and Methods: In order to produce sterile C. roseus seedlings and to prepare explants, seeds cultured on MS culture medium. The effect of the type of culture medium (MS, 1/2 MS and B5), the type of explant (leaf and stem), the type of bacterial strain (A4, 1132 and 2656) and the type of plant genotype (white and red) on the amount of hairy root induction in a completely randomized design was investigated. Extraction of samples was done using methanol solvent, then the amount of indole alkaloids was calculated using HPLC method and standard curve. The nature of transgenic hairy roots was confirmed after DNA extraction from different samples using PCR method with specific primers (rolB and virD).
Results: According to the results, the most efficient strain in transferring T-DNA to the explants and the appearance of hairy roots was related to A4 strain (53.8%). 1/2 MS culture medium with 77.69% of root induction was the best medium combination for the appearance of hairy roots. Also, white genotype (62% rooting) and leaf explant (77% rooting) were identified as the best genotype and explant for the production of hairy roots. After the specific amplification of the rolB gene and the lack of amplification of the virD gene in the PCR reaction in the samples, the molecular confirmation of the transgenic nature of the hairy root clones was done. Based on the results of HPLC experiment, the average amount of vindoline and catharanthine in hairy roots (0.24 and 0.615 mg/g dry weight) is higher than aerial parts (0.106 and 0.488 mg/g dry weight) and normal root (0.021 and 0.013 mg/g dry weight). The amount of these alkaloids in the aerial parts was higher than in the normal roots of the plant.
Conclusion: The production of hairy roots in response to the inoculation of plant explants with A. rhizogenes is influenced by various factors such as the type of explant, the genotype of the plant, the strain of bacteria and the composition of culture medium. These factors must be optimized to establish effective hairy root cultur in a plant. It seems that with the introduction of rol genes from bacteria to the plant genome (hairy root) and as a result of the change in the amount of endogenous hormone production and the response of the plant cell to the entry of bacterial genes, the amount and profile of the production of the secondary metabolites of the plant will change. As a result, by selecting clones with increased production of medicinally important alkaloids and optimizing the conditions of large scale production of secondary metabolites in bioreactors, we can move towards the commercial production of these compounds through cell and plant tissue culture.

Abstract Aim: Cartilage tissue has limited capacity for spontaneous repair and self-renew due to the absence of vascularization and progenitor cells, thus requiring a therapeutic approach to repair tissue damage. Recently, extracellular vesicles (EVs) have attracted attentions because of their major roles in cell communication and tissue repair by regulating cellular processes such as cell growth, differentiation, and proliferation. Therefore, it is necessary to isolate extracellular vesicles with chondrogenic potential from an appropriate cellular source for cartilage regeneration. This study aims to compare the chondrogenic ability of extracellular vesicles derived from cocultured -chondrocytes / mesenchymal stem cells (CHO / MSC) at ratios of 1/2 and 1/4.
Material and Methods: Towards this goal, chondrocytes and mesenchymal stem cells were isolated from rabbit articular cartilage and bone marrow, respectively. Mesenchymal phenotype of isolated MSCs were characterized based on their surface markers and by differentiation into mesenchymal lineages. Chondrocytes and mesenchymal stem cells were co-cultured with defined ratios, their conditioned media were collected and extracellular vesicles were extracted with an ultracentrifuge. Concentrations of extracellular vesicles were determined using BCA Protein Assay Kit, then EVs were characterized in terms of size, morphology, and expression of surface markers by dynamic light scattering (DLS), scanning electron microscope (SEM) and western blotting, respectively. Mesenchymal stem cells were treated with different concentrations of extracellular vesicles (50, 100, and 150 ug/ml) for 21 days. Quantitative real time-PCR (qRT-PCR) and histological analysis were subsequently performed to assess the quality of chondrogenic differentiation among experimental groups. The differentiation of MSCs to osteogenic and adipogenic lineages  was demonstrated by Oil Red and Alizarin Red staining after 21 days.
Results: The results of flow cytometry showed that CD90 was expressed by 88.6% of the cell population as a positive marker and CD34 as a negative marker was only expressed in 5.48% of the cell population. SEM micrographs confirmed the spherical shape of CHO/ MSC-EV 1/2 and CHO/MSC-EV 1/4 and the mean particle size of isolated EVs were 51.66 ± 8.15 and 19.11 ± 6.03 nm, respectively. Special surface markers for extracellular vesicles containing CD9 and CD81 were expressed in both groups. At concentrations of 100 and 150 ug/ml of CHO/MSC-EV 1/2, higher cartilage - specific markers, including Col II were observed compared to CHO/MSC-EV 1/4. Similarly, safranin O and toluidine blue staining revealed the more deposition of aminoglycan and proteoglycan at concentrations of 100 and 150 ug/ml of CHO/ MSC-EV 1/2, compared to CHO/ MSC-EV 1/4. This study demonstrated the chondrogenesis potential of extracellular vesicles, especially in CHO /MSC-EV 1/2 and extracellular vesicles derived from co-culturing chondrocytes/MSCs improve matrix production and chondrogenesis.
Conclusion: Our research shows that chondrocyte-MSCs proximity is essential for the response as improved viability, chondrogenesis, and matrix formation in recipient MSCs. It is also proposed that co-cultured derived EVs with the ability to promote chondrogenesis have the potential to be utilized in cartilage regeneration.  Due to their immunogenicity and their low tumorigenic potential, extracellular vesicles can be used to help repair cartilage tissue.

Abstract Aim: Potato (Solanum tuberosum L.) is a crop belongs to the Solanaceae family. It is the fourth strategic crops following wheat, rice, and corn. Salicylic acid or orthohydroxybenzoic acid, act as a regulator that can regulate physiological, biochemical and signaling functions in plant metabolic processes. Aim: Salicylic acid as a growth regulator can interact with auxin and gibberellin hormones and effectively induce growth and metabolic processes in potato plants. Potato is one of the most important agricultural plants and in vitro culture can be used as a model in laboratory conditions. The aim of this study is to investigate the effect of salicylic acid on growth indicators and its interaction with other hormones.
Material and method: In this study, potato seedlings with uniform growth were grown on MS culture medium with concentrations of zero, 0.1, 10 and 100 μM salicylic acid. The seedlings were placed in the growth room under light conditions of 16 hours of light and 8 hours of darkness at room temperature. After four weeks, the grown seedlings were used for wet weight, dry weight, shoot and root length, photosynthetic pigments, auxin and gibberellin hormones content were measured. The statistical analysis was performed using SPSS software.
Results: According to the data, growth parameters in the samples treated with salicylic acid showed similar patter. Our results showed that relative water capacity, root and shoot length and pigments under 0.1, 1 and 10 μM significantly increased compared to control samples. While at the concentration of 100 μM, a decreasing trend was observed in the growth parameters and showed a significant decrease compared to the control samples. In this study, the maximum values ​​in all growth parameters were obtained under the concentration of 10 μM salicylic acid. Furthermore, based on the results, the concentration of 0.1 μM salicylic acid had no significant difference on gibberellin hormone content. While the gibberellin hormone content under salicylic acid treatment at concentrations of 1 and 10 μM significantly increased compared to the control sample, and the maximum level was observed at 10 μM. The concentration of 100 μM, unlike the other concentrations, showed a significant difference compared to the control sample with a decreasing trend. Also the results in auxin levels showed a similar pattern with growth parameters and gibberellin hormone. The auxin levels showed a significant increase under the concentrations of 1, 10 and 100 μM compared to the control sample, and the maximum levels were observed at the concentration of 10 μM. However, concentrations of 1 and 10 μM did not show significant differences. Similar to the results of gibberellin hormone, auxin hormone showed a significant difference under the concentration of 100 μM with a decreasing trend compared to the control sample.
 Conclusion: It seems that salicylic acid increased in low concentrations due to the direct effects on the metabolic and enzymatic processes of growth parameters, photosynthetic pigments, and auxin and gibberellin content. On the other hand, at high concentrations, the growth parameters were inhibited due to the high accumulation of reactive oxygen species produced by salicylic acid and the resulting oxidative stress.
 

Abstract

Abstract Aim: we aimed at presenting a simple and efficient method for isolating and characterizing the neural crest cells.
Material and Methods: The hen’s fertilized eggs were incubated for about 35h at 38°c and 55-60% humidity until the embryos reached to stages 10-12 according to Hamburger-Hamilton developmental stage table. Then the embryos were removed from the egg’s yolk and the neural tube was isolated and cultured for 24 h in a tissue culture dish to release neural crest cell. Then after, the neural tube was removed and allowed to NCC to expand for further 5 days. Finally, the cells were collected and subjected to PCR to study their gene expression profile.
Results: The neural tube released NCC and these cells proliferated in culture condition. They also expressed markers including Slug, Sox9 ,and Sox10 by the RT-PCR method.
Conclusion: The neural tube can release NCC in culture condition and these cells can proliferate in the presence of an appropriate medium.
 

Abstract Aim: Low back pain (LBP) is one of the most common musculoskeletal diseases in the world. Apoptosis of nucleus pulposus (NP) cells and structural changes in the intervertebral disc (IVD) matrix cause its degeneration and are directly related to LBP. Because of exosomes derived from mesenchymal stem cells (MSC-Exo) are high therapeutic potential and may be valuable options for decreasing intervertebral disc degeneration (IDD). 
The present study evaluates changes in the expression level of genes responsible for repairing NP cells (collagen and aggrecan) and tumor necrosis factor-alpha (TNF-α) gene and the effectiveness of exosomes in inhibiting excessive apoptosis of NP cells under inflammation.
Material and Methods: Exosomes were separated by ultracentrifuge. They were identified with the help of transmission electron microscopy (TEM), atomic force microscope (AFM), and dynamic light scattering (DLS). Cell viability was evaluated by MTT assay and DAPI staining. Real-time PCR measured the expression of collagen, aggrecan, and TNF-α genes.
Results: Exosomes are vesicles with a diameter of about 35.5 to 100 nm. Based on the findings of the MTT assay, the survival rate of the inflammatory cells treated with exosomes was significantly different from the inflammation group without treatment (*P<0.05 at the dose of 100 μg/ml, **P<0.01 at the doses of 25 and 50 μg/ml). DAPI results showed a decrease in cell death in the exosome-treated group. Real-time PCR results showed increased expression of collagen (*P<0.05) and aggrecan (***P<0.001) genes and decreased the TNF-α (**P<0.01) gene in inflammatory cells treated with exosomes.
Conclusion: Exosomes derived from mesenchymal stem cells can increase collagen and aggrecan gene expression and decrease TNF-α gene expression in treated cells.

Abstract Introduction: Homeodomain transcription factor 1 (PDX-1) is necessary for proper pancreatic development and β-cell function. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, in conjunction with CRISPR-associated protein 9 (Cas9), facilitates modifications, particularly deletions, within the genome. The use of lentiviral vectors for gene transfer to somatic stem cells has increased significantly. They harbor advantageous features, including a relatively high gene loading capacity, a diminished likelihood of gene expression silencing following integration into the genome, and high efficiency in delivering genes to a variety of target cells. Furthermore, they elicit no undesirable immune reactions and offer a broad selection of alternative envelope proteins.
Aim: This study aims to introduce a mutation in the chicken PDX-1 gene. A guide RNA (gRNA) will be designed to complement a specific region of the PDX-1 gene. This approach aims to create targeted mutations with potential therapeutic applications in gene therapy.
 
Materials and Methods: The PDX-1 gene was cloned using the LentiCRISPRv2GFP vector. To delete and modify the PDX-1 gene, a specific single guide RNA (sgRNA) sequence is designed. This sgRNA sequence is approximately 100 base pairs (bp) in length, with about 76 bp derived from the scaffold segment already integrated into the LentiCRISPRv2GFP vector.
The region responsible for binding the sgRNA to the target genomic sequence is 20 nucleotides long and is complementary to 20 nucleotides in exon one of the PDX-1 gene. The CRISPR Design Tool (http://crispr.mit.edu) was used to design a specific sgRNA sequence targeting the PDX1 locus (sgPDX1).
The double-stranded oligonucleotide fragment and the LentiCRISPRv2GFP vector were subjected to enzymatic digestion using the BsmbI restriction enzyme. To create the LentiCRISPRv2GFP-sgPDX1 vector, the cleaved sgPDX1 sequence was joined with the cleaved vector. This was accomplished by mixing the vector and nucleotide fragment and subjecting them to a ligation reaction using the T4 ligase enzyme. The resulting vector contains a transcript of the sgRNA, specifically sgPDX1.
The heat shock method was employed to transform Escherichia coli Top10 host cells with the cloned LentiCRISPRv2GFP-sgPDX1 vector. To confirm the presence of the sgPDX-1 gene in the vector, a polymerase chain reaction (PCR) was conducted using primers targeted at a portion of the U6 promoter sequence (forward primer) and a segment downstream of the sgRNA gene region (reverse primer). Additionally, each PCR product was analyzed using enzymatic digestion with EcoRV and BsmBI enzymes. To confirm the successful cloning, the PCR product was sequenced.
Results: The PCR results showed an amplified fragment measuring 267 base pairs (bp), which indicated the presence of the sgPDX-1 gene. This was visualized on an agarose gel. Additionally, the reverse digestion with two restriction enzymes produced a 2000 bp fragment isolated from the vector. The sequencing results confirmed that the cloning process was successful.
Discussion: Genome editing technology has been highlighted in bird species due to their significant potential for economic production and creating models for biological research. The application of CRISPR gene editing technology in avian research has yielded promising advancements, particularly in the modification of somatic cells and tissues in birds. By precisely targeting primordial germ cells (PGCs), researchers can implement specific genetic modifications within the chick genome, thereby facilitating the development of genetically edited chicks. Numerous studies have investigated the role of the PDX-1 gene in gene therapy using the CRISPR/Cas9 system across various organisms. However, this study is the first to design and produce a recombinant construct containing the chicken PDX1 gene using a lentiviral vector with the CRISPR/Cas9 construct. This advancement aims to create a practical research platform for future studies by enabling the direct transfer of this recombinant construct into chick embryos and cells.
Conclusion: The chicken PDX-1 gene was cloned into a lentiviral vector containing the CRISPR/Cas9 construct. The recombinant vector LentiCRISPRv2GFP-sgPDX1 was successfully obtained. The gene construct developed in this study has significant potential for investigating gene function and for use in gene therapy. Consequently, utilizing this construct may enable the creation of an animal model in chicken embryos featuring defects, including an empty organ or tissue cavity. Such a model will allow for the investigation and treatment of the defects using specific drugs and cell lines.

Abstract Aim: Turmeric contains a high amount of curcumin (Cur), which exhibits strong antioxidant activity. Cur binds to antioxidant response elements for gene, thus suppressing reactive oxygen species (ROS). Reactive oxygen species are short-lived, highly electrophilic molecules. When intracellular antioxidants are reduced or ROS accumulate excessively, it brings about the imbalance in the redox state which leading to oxidative stress. DEHP consists of a pair of eight-carbon esters linked to a benzene-dicarboxylic acid ring with chemical formula of C24H38O4. DEHP is used as a plasticizer in many polyvinyl chloride products, especially in medical devices such as intravenous bags and tubing, umbilical artery catheters, blood bags, infusion tubing, enteral nutrition feeding bags, nasogastric tubes and dialysis bags. Due to non-covalent binding to the plastic products, DEHP leaches into biological fluids during medical treatment causing contamination. There is ample evidence that DEHP induces oxidative stress and causes apoptosis in cells. Therefore, the study aimed to investigate the effects of Cur on DEHP-induced oxidative stress in bone marrow mesenchymal stem cells (BMSCs), as DEHP has been found to induce oxidative stress in BMSCs.
 Materialand Methods: Rat BMSCs was extracted and after the 3rd passage, their cell viability and proliferation were examined in the presence of various concentrations (0.05,0.1, 0.25, 1, 2.5 and 5 μM) of Cur for a period of 4 days. Subsequently, a concentration of 0.1 μM Cur was chosen for further assessment of viability and proliferation abilities (measured by population doubling number (PDN)), total protein, malondialdehyde (MDA), total antioxidant capacity (TAC), catalase and superoxide dismutase (SOD) activity as well as the expression of NFkB and Nrf2 both individually and in conjunction with 100 or 500 μM of DEHP (chosen based on previous study) for durations of 4 and8 days. The data underwent statistical analysis with P<.05 considered as the minimum level of significance.
 Results: Concentrations of 0.05, 0.1, and 0.25 μM of Cur were found to have no effect on the viability (P<0.05) of the BMSCs but significantly increased proliferation ability (P<0.0001). Conversely, concentrations of 1, 2.5, and 5 μM of Cur significantly reduced viability (P<0.0001). The concentration of 0.1 μM was chosen for further analysis based on viability tests and PDN analysis. This selected concentration notably increased BMSCs proliferation after 4 days, while higher concentrations (1, 2.5, and 5 μM) reduced both viability and proliferation. Furthermore, this concentration counteracted the effects of DEHP at both day 4 and day 8 by reducing MDA levels and increasing TAC levels as well as catalase and SOD activity - particularly at day 8 compared to the DEHP-treated groups. Additionally, Cur was found to reduce NFkB expression while increasing Nrf2 expression in BMSCs.
Conclusion: Curcumin mitigated oxidative stress induced by DEHP through the Nrf/NFkB pathway. Moreover, it enhanced the cell viability and proliferation capacity of BMSCs in the presence of DEHP. Therefore, patients undergoing hemodialysis and blood transfusion, who may be exposed to DEHP from medical devices like tubes and other plastic products, should consider consuming turmeric to counteract the oxidative impact of this chemical.

Abstract Aim: This research aimed to study the anticancer effects of nano-selenium oxide-enriched Saccharomyces bullardi on DMBA-induced breast cancer cells in rats.
Material and Methods: In order to induce cancer, the carcinogen with a concentration of 60 mg/kg was injected into the left breast nipple of the female rats. When the tumors grew to about 10 mm, the cancerous tumor was isolated from the breast of the animals and used to make tissue sections and individual cells. Cancer cells were treated with different concentrations of S. boulardi suspension and nano-selenium oxide-enriched S. boulardi. The cytotoxicity assay was performed with two methods of MTT and Trypan blue staining.
Results: Comparison between two cell groups treated with S. boulardii and nano-selenium oxide-enriched S. boulardi using two tests, it was found that there were significant changes in cancer cells viability in both 0.5,1 µg/ml concentrations. with increasing the concentration, the cancer cell viability was significantly decreased. cell viability in the cell line treated with nano-selenium oxide-enriched S. boulardi yeast, with the increase in the concentration of enriched yeast was decreased. A comparison between the yeast and enriched yeast in different concentrations, revealed that there was a significant difference between cytotoxicity effects of them on the cells.
Conclusion: Probiotic yeasts could be an appropriate candidate for the treatment of some diseases including cancers, especially when combined with selenium compounds.
 

Abstract

Abstract Aim: In thyme, thymol and carvacrol have attracted the attention of pharmacologists due to their various therapeutic properties such as anti-tumor activity. To date, a variety of treatments such as elicitors have been proposed to increase the content of thymol and carvacrol. This study was also conducted with the aim of evaluating the synergistic effect of UV-A ray and methyl jasmonate on the biosynthesis of thymol and carvacrol.
Material and Methods: Thyme seeds were planted in plastic pots at a factorial experiment in time with a completely randomized design with three replications under greenhouse conditions. The five-leaf seedlings were separately and simultaneously irradiated with UV-A and sprayed with 0.1 mM methyl jasmonate. After 24 and 48 h of treatment, the expression levels of GTS, DXR, CYP180, and CYP178 genes were measured by Real-Time PCR and the metabolite levels were measured by HPLC.
Results: The expression of GTS, DXR, CYP180, and CYP178 genes was affected by the synergistic impact of methyl jasmonate and UV-A. The transcript level of these genes increased significantly after 24 h of individual treatments and this increase was more in the combined treatment of hormone and radiation. However, the expression of these genes was linked with a significant decrease after 48 h of treatment. The increasing and decreasing trend of thymol and carvacrol content in the elicitor treatments was also in accordance with the expression of studied genes.
Conclusion: Our observations suggest that thyme uses synergistic signaling to increase its metabolite levels during the first 24 h of hormone and radiation treatments. And after that, other defense mechanisms are activated and then the level of secondary metabolites decreases.

Abstract Introduction: Numerous studies on microRNAs (miRNAs) have indicated that abnormal interactions between miRNAs and mRNAs contribute to the development and worsening of endometriosis. miR-223 influences genes associated with various biological functions such as signaling pathways, gene expression, cellular development, proliferation, angiogenesis, and programmed cell death. Alterations in genes have been demonstrated to be significant in the progression of endometriosis. The p53 gene, recognized as a tumor suppressor, plays a vital role in inhibiting cancer by regulating cell growth and division. Recent investigations suggest a potential connection between the p53 gene and endometriosis. In addition, Forkhead box protein O1 (FOXO1) acts as a cell-specific core transcription factor essential for effective endometrial remodeling throughout the menstrual cycle and may play an important role in the onset of endometriosis.
Aims: This study aimed to explore the expression levels of miR-223-3p and its target genes, TP53 and FOXO1, in cases of ovarian endometriosis through bioinformatic analysis and experimental verification.
Materials and methods: Gene expression data were obtained from the GSE105765 dataset in the Gene Expression Omnibus (GEO) database, which encompasses both normal (eutopic) and ectopic tissue samples from endometriosis patients. Differentially, expressed genes were pinpointed using the "limma" package in R software, with the results visualized as a heat map. Among the various expressed miRNAs, miR-223-3p was chosen for further investigation. Subsequently, the target genes of miR-223-3p were explored utilizing the miRDB database, leading to the identification of FOXO1 and TP53 as target genes. In the experimental component of this study, 40 ectopic tissue samples, 40 eutopic tissue samples from individuals with endometriosis, and 40 normal endometrial samples (control group) were analyzed. Following the extraction of total RNA and synthesis of cDNA, the expression levels of miR-223-3p, TP53, and FOXO1 were assessed using Real-Time PCR.
Results: The average relative expression of miR-223-3p in the ectopic group was measured at 4.13±0.72, while in the eutopic group it was 1.10±0.10, and in the control group it was 1.00±0.09. The relative expression of miR-223-3p was significantly higher in the ectopic group compared to both the eutopic and control groups (p<0.0001), whereas no significant differences were found between the control and eutopic groups (p=0.54). The average expression of FOXO1 in the ectopic group (0.47±0.12) was significantly lower than that in the eutopic group (1.02±0.06) and the control group (1.001±0.11). One-way ANOVA revealed a significant reduction in FOXO1 expression in the ectopic group when compared to the eutopic group and the control group (p<0.0001). However, there was no significant difference in FOXO1 expression between the eutopic and control groups (p=0.53). The relative expression level of the TP53 gene was analyzed in the ectopic group (0.335±0.14), the eutopic group (1.05±0.09), and the control group (1.01±0.12). Statistical evaluations showed that TP53 gene expression was significantly lower in the ectopic group compared to both the eutopic and control groups (p<0.0001). Conversely, no significant difference was observed in TP53 expression between the eutopic and control groups (p=0.28).
Conclusion: The findings indicate that miR-223-3p expression in ectopic samples was significantly elevated compared to both the eutopic and the control samples, while the expression of the FOXO1 and TP53 genes in the ectopic group was notably reduced relative to the eutopic and control groups. These results suggest that the expressions of miR-223-3p, TP53, and FOXO1 genes are changed in the endometrium of individuals with endometriosis, highlighting their potential roles in the disease's pathogenesis and their therapeutic implications.

Abstract Aim: This study aimed to compare the expression levels of LncRNA CRNDE in malignant colorectal tumorsand adjacent normal tissues of patients by Real Time-PCR.
Material and Methods: 20 pairs of colorectal tumors and adjacent normal tissue specimens were prepared from the tumor bank of Imam Khomeini Hospital in Tehran University of Medical Sciences. After total RNA extraction from cancerous and normal tissues, cDNA was synthesized, and the expression levels of LncRNA CRNDE were examined using the Real Time-PCR method. A one-way analysis of variance (ANOVA) and Tukey's HSD test used for statistical analysis of results.
Results: The results showed a two-fold increase in LncRNA CRNDE expression in all 20 colorectal cancer samples compared with the normal cells.
Conclusion: According to the significant increase of the LncRNA CRNDE expression in cancer cells relative to normal cells and its presence in the bloodstream, this LncRNA maybe used as a non-invasive diagnostic biomarker for early detection of colorectal cancer.
 

Abstract Aim: As the second leading cause of death worldwide, cancer has been a long-standing and rapidly evolving focus of biomedical research and practice. Ovarian cancer is one of the deadly malignancies of women, which is known as the "silent killer". Because its symptoms usually appear when the disease has reached advanced stages and is mostly incurable. On the other hand, currently common treatment methods are associated with various limitations, failures and side effects, which have made researchers pay more attention to the compounds extracted from plants as anti-tumor and anti-cancer agents in the last two decades. The studies conducted on the various properties of Ashwagandha (Withanaia somnifera) show that this plant has therapeutic effects and anti-cancer properties that can even be effective on the process of apoptosis mediated by different genes, including P53. Today, P53 is known as a gene It has a key role in all types of cancers and mutations in this gene have been observed in 60% of ovarian cancers. Therefore, the aim of this study is to investigate the cytotoxic effects of the hydroalcoholic extract of Ashwagandha (Withanaia somnifera) and the changes in P53 gene expression in response to this substance in ovarian cancer cells (cell line A2780).
Material and method: For this purpose, extraction of Ashwagandha plant was done using soaking method. Then, the compounds present in the extract were determined using standard phytochemical tests. Then A2780 cells were treated with different concentrations of Ashwagandha extract for 24, 48 and 72 hours. The effects of this extract on cell survival were evaluated using the MTT test, and IC50 was calculated. Then, A2780 cells were exposed to concentrations of 250 and 500 µg/ml for 24 and 48 hours, and the level of P53 gene expression was investigated by Real-Time PCR. Finally, statistical analysis and data interpretation was done using GraphPad Prism software.
Results: MTT results showed that Ashwagandha extract at concentrations of 62.5, 125, 250, 500, and 750 µg/ml significantly decreased cell viability in a time- and concentration-dependent manner. The lowest percentage of survival rate is related to the concentration of 750 μg/ml and the time of 72 hours, while the highest percentage is related to the concentration of 62.5 μg/ml and the time of 24 hours. After 24, 48, and 72 hours, respectively, IC₅₀ was obtained at concentrations of 512.6, 339, and 226.6 μg/ml, and Real-Time PCR results showed that the expression of the P53 gene during 24 hours of treatment with concentrations of 250 and 500 μg/ml of extract had increased significantly. Also, increasing the concentration of the extract had a significant effect on the expression of this gene.
Conclusion: The results of this study indicate the cytotoxic effect of Ashwagandha extract on ovarian cancer cells, and by increasing the expression of the P53 gene, it can induce anti-cancer effects. It is hoped that by knowing the mechanisms of action of this medicinal plant and designing new drugs, it will be possible to stop the progression of ovarian cancer and thus help to increase the life span of these patients against this silent killer.

Abstract Aim: Cancer is one of the most important diseases of this century, which affects many people all over the world, and while the prevalence of this disease is increasing, no suitable treatment has been found yet. In the meantime, breast cancer is the most common cancer and the leading cause of cancer-related death in women worldwide. In the search for cancer treatment solutions, researchers synthesize numerous medicinal compounds for various cancers and study their effects. Sorafenib is a urea multikinase inhibitor which causes apoptosis and inhibits angiogenesis and cancer cell proliferation. This compound is currently used as a treatment for hepatocellular cancer, and tests have also been carried out to verify its performance in treating other cancers. Sorafenib derivatives were also synthesized and analyzed. In this research, the anticancer effect of one of the derivatives of sorafenib named (2E,2´E)-2,2´-(1,4-phenylene bis(methanylidene)bis(N-(4-chloro-3-(tri) Fluoroethyl (phenyl) hydrazine carboxamide, abbreviated as SO-D-3, was evaluated against three breast cancer cell lines.
Materials and methods: Three breast cancer cell lines including MCF7, 4T1 and MDA were prepared and cultured in RMPI culture medium. Cells were treated with concentrations of 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM of SO-D-3, and their survival rates were measured using the MTT test during 24, 48 and 72 hours. The induction of apoptosis by SO-D-3 in the studied cancer cells was investigated by flow cytometry. In addition, the level of Hsp70 and Casp8 proteins as implicated in apoptosis were investigated using the western blot method. The results were statistically analyzed using SPSS software.
Results: The results of the MTT test showed that the treatment of cancer cells significantly decreases the survival of these cells compared to the control. The IC50 for MDA-MB-231 cells was significantly lower than MCF7 and 4T1 cells at 24 hours (157.4 µM for MDA-MB-231 compared to 843.8 µM for MCF7 and 1212 µM for 4T1). However, the difference decreased with increasing treatment time (77.47 μM and 23.38 μM for MDA-MB-231, 107.3 μM and 36.69 μM for MCF7, and 83.63 μM and 16.58 μM for 4T1 at 48 hours and 72 hours respectively). Flow cytometry analysis revealed that in cells treated with SO-D-3, apoptotic cells increased significantly. Examination of the protein level using the western blot technique also showed that the HSP70 protein was significantly decreased in MCF7 and 4T1 cells. On the other hand, the level of Casp8 protein was significantly increased in 4T1 and MDA cells.
Conclusion: The results of the present study showed that SO-D-3 can cause the death of breast cancer cells in a time-dependent manner. Subsequent experiments showed that SO-D-3 exerts this action through the induction of apoptosis. Therefore, according to the results of the present research, SO-D-3 can be introduced as a compound with potential anti-cancer properties that can cause cell death by reducing Hsp70 and changing the expression of Casp8 involved in the external pathway of apoptosis.

Abstract Aim: Breast cancer remains a significant health challenge, being one of the most common cancers among women and a leading cause of cancer-related deaths globally. The need for effective treatment options is paramount, and recent research has spotlighted sulfonamide derivatives for their notable antitumor properties. These compounds have shown considerable cytotoxic effects against various cancer cell lines, including breast cancer, through mechanisms such as apoptosis induction and inhibition of heat shock protein 70 (HSP70), which is crucial for cancer cell survival. This study focuses on synthesizing a solid lipid nanocarrier that incorporates a sulfonamide derivative, aiming to enhance its anticancer efficacy against the MCF-7 breast cancer cell line. The encapsulation of sulfonamide within these nanoparticles is expected to improve its bioavailability by increasing solubility and stability, thereby allowing higher concentrations to effectively reach tumor sites. Additionally, these nanoparticles are designed to minimize systemic toxicity and protect healthy tissues from adverse effects, resulting in enhanced therapeutic efficacy.
methods: The research involved synthesizing solid lipid nanoparticles (SLNs) containing the sulfonamide derivative, followed by a comprehensive analysis of their physicochemical properties. Key parameters assessed included drug loading capacity, morphology, size distribution, and release profile using techniques such as Fourier Transform Infrared Spectroscopy (FTIR), Dynamic Light Scattering (DLS), Atomic Force Microscopy (AFM), and dialysis methods. The cytotoxicity of the nanocarrier was evaluated on MCF-7 cells through the MTT assay, alongside assessments of cell proliferation and migration using scratch assays.
Results: The synthesized nanocarrier had an average size of 315 nanometers with a Polydispersity Index (PDI) of 0.3, indicating good uniformity. AFM analysis confirmed its spherical morphology. The nanocarrier demonstrated a high drug loading capacity of approximately 82% for the sulfonamide derivative and facilitated a controlled release over time. Cytotoxicity assays revealed that while the blank nanocarrier was safe, the formulation containing the sulfonamide derivative exhibited significantly enhanced cytotoxic effects on MCF-7 cells at lower concentrations compared to the free sulfonamide alone. This improvement in efficacy is attributed to the controlled release mechanism that allows for sustained exposure of cancer cells to the active compound. Moreover, the nanocarrier was more effective in inhibiting both growth and migration of cancer cells than treatments with free sulfonamide, as evidenced by scratch assays.
Conclusion: This research highlights the potential of solid lipid nanoparticles in improving the therapeutic effectiveness of sulfonamide derivatives against breast cancer. The synthesized nanocarrier demonstrates favorable physicochemical properties, including high drug loading capacity and controlled release, which are essential for effective treatment outcomes. Notably, encapsulating sulfonamide derivatives significantly enhances cytotoxicity against MCF-7 cells at lower doses compared to their unencapsulated counterparts, emphasizing its promise for improved therapeutic strategies. Furthermore, these nanoparticles effectively inhibit cell proliferation and migration, contributing to tumor growth suppression. This innovative approach aims not only to enhance treatment efficacy but also to minimize systemic toxicity and protect healthy tissues from adverse effects. Future research should focus on innovative clinical applications and further optimization of these nanocarriers to maximize their potential in oncology, paving the way for more effective treatments for breast cancer.

Abstract

Abstract Aim: This experiment was performed to investigate the effect of ethanolic extract of yarrow leaves and flowers on the survival of three cancer cell lines namely; HELA, CAOV, MCF7, for the possibile production of inexpensive drugs without side effects.
Material and Methods: After incubation of cancer cell liners 48 hours, cell suspension was prepared and distributed with cells at different concentration in culture medium for 24 hours. Then Achilleawilhelmsii leaf and flower extract were prepared and sterilized. Consecutive dilutions of 0.4, 0.8, 1.6, 3.2 and 6.4 mg / ml were prepared and added to each well containing cell suspension and placed in an incubator. The plates were examined microscopically after 48 hours and light absorption was determined after MTT assay.
Results: The results showed that in MCF7 with 0.4 mg / ml; CAOV, and HELA cancer cell lines, the lowest cell survival rate was observed at the concentration of 6.4 mg / ml leaf extraction. In comparison, the highest anti-cancer effect of yarrow flower extract on three cancer cell lines was recorded in MCF7 cancer cells at a concentration of 6.4 mg / ml, while in HELA cancer cell line the lowest cell survival ware observed at the concentration of 6.4 mg / ml in a 72-hour treatment.
Conclusion: According to the results, yarrow flower and leaf extract treatment is recommended to decrease of three cancer cell lines survival.
 
 

Abstract Aim: Inflammation is a protective physiologic response against pathogens, trauma and injury (1, 2). Inflammation pathway are different in human body and some main pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α have key role in pathogenesis of inflammation (3). Current anti-inflammatory drugs including corticosteroids and non-steroid anti-inflammatory drugs have serious side effects (6), so, research on new compounds with anti-inflammatory property is necessary. Astaxanthin is a natural carotenoid with various pharmacological effects. Haematococcus Algae is one of the marine sources of astaxanthin. Anti-inflammatory and antioxidant effects of astaxanthin were reported in previous studies (8-10), but the molecular mechanism of the anti-inflammatory effect of astaxanthin on the peripheral cells of the immune system is not clear. The aim of this study was to evaluate anti-inflammatory effects of astaxanthin on TLR4 gene expression and secretion of key inflammatory cytokines including IL-1, IL-6, and TNFα in RAW264.7 macrophage cells.
Methods and Material: The RAW264.7 macrophage cells was used in this study. Astaxanthin was extracted from Haematococcus Algae, then different doses of 25, 50, 100 μM of astaxanthin were used to treat the cells. Cells divided to three groups: one group received LPS (1µg/ml), Treatment group received LPS (1µg/ml) in combination to different dose of astaxanthin (25, 50, 100 μM) and control group without LPS astaxanthin. After 24 hour incubation, cell viability was assay with MMT test.TLR4 gene expression was measured by RT-PCR at 24 and 48 hours after induction with LPS. β-actin was considered as housekeeping gene. The levels of IL-1β, IL-6, and TNFα cytokines were measured with a commercial sandwich ELISA kit. The optic absorbance was read at 45 nm. Descriptive statistics and one-way ANOVA test were used to analyze the data. To analysis the fold change of gene expression, formula 2 -^ΔΔCT   was used.
Results: None of different doses of astaxanthin (25, 50, 100 μM) and LPS (1µg/ml) had no toxicity effect on macrophages cell. (P>0.05). LPS increased TLR 4 gene expression at both 24 and 48 hours after induction, (P <0.001) in compare to control group. 

Abstract Introduction: Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder among women of reproductive age. It is characterized by hormonal imbalances, irregular menstrual cycles, hyperandrogenism, and insulin resistance. The prevalence of this condition is on the rise, largely due to changes in lifestyle, poor dietary habits, and increasing rates of obesity. PCOS not only affects reproductive health but also poses long-term metabolic risks, including type 2 diabetes, cardiovascular disease, and dyslipidemia. Despite its high prevalence, the exact cause of PCOS remains unclear, and its heterogeneous nature makes diagnosis and management challenging. Early identification and a multidisciplinary approach to treatment are essential for improving patient outcomes and preventing long-term complications. Aim: Given the prevalence of polycystic ovary syndrome and its importance as a major problem affecting women's health and fertility, the present study aimed to investigate the relationship between anti-mullerian hormone with insulin resistance and reproductive hormones in women with this syndrome. Materials and Methods: A total of 100 women aged 18 to 45 years, including both healthy individuals and those diagnosed with PCOS, participated in this study. The diagnosis of PCOS was confirmed by a gynecologist based on clinical examination and transvaginal or pelvic ultrasound findings. For all participants, the following parameters were recorded: age, weight, height, waist circumference, hip circumference, fasting blood glucose, insulin levels, and serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estrogen, progesterone, free testosterone, and anti-Müllerian hormone (AMH). Results: There was no significant difference in mean age and FSH levels between the experimental groups. However, women with PCOS showed a significant decrease in estradiol and progesterone levels, along with a significant increase in body mass index (BMI), LH, free testosterone, AMH, and insulin resistance compared to the control group. Pearson correlation analysis revealed no significant relationship between AMH and the hormones LH, FSH, testosterone, or progesterone in either the control group or the PCOS group. However, a significant positive correlation between AMH and estradiol was observed in the control group, while a significant negative correlation was found in the PCOS group. Discussion: Overweight, obesity, and hormonal imbalances are common characteristics observed in women with PCOS. In the control group, a positive and significant correlation between serum AMH and estradiol levels suggests a normal physiological relationship between follicular activity and estrogen production. In contrast, the significant negative correlation observed in the PCOS group indicates a disruption in the hypothalamic-pituitary-ovarian (HPO) axis and reflects abnormal folliculogenesis. This dysregulation may lead to anovulation and persistent immature follicles, which are hallmarks of PCOS. Moreover, elevated AMH levels in PCOS patients are often linked with increased ovarian follicle count, yet without proper maturation, resulting in hormonal imbalance and infertility. These findings highlight the importance of early detection and hormonal monitoring in women with PCOS to prevent long-term reproductive and metabolic complications. Conclusion: These findings underscore the potential utility of AMH–estradiol correlation patterns as a supplementary diagnostic marker for PCOS. Given the complex and heterogeneous nature of PCOS, the identification of reliable biomarkers is crucial for early diagnosis and tailored treatment strategies. Further studies with larger sample sizes and diverse populations are needed to validate the role of AMH in PCOS diagnosis and its relationship with estradiol levels. Moreover, exploring the underlying mechanisms of AMH dysregulation in PCOS could provide valuable insights into potential therapeutic targets for managing both reproductive and metabolic complications associated with the condition.

Abstract Aim: Infertility is a life crisis that affects patients worldwide. Infertility is defined as failure to conceive after 12 months of sexual activity, affecting 15-17% of couples worldwide. and about 50% of them are related to the factors of female infertility. Activating the process of meiosis in the oocyte and its maturation has been one of the important therapeutic goals of infertility researchers. Today, inducing oocyte growth and development outside the body is one of the methods used in assisted reproduction technology. Bone marrow is a complex organ in which different lineages of hematopoietic and stromal cells support hematopoiesis. Extracellular vesicles (EVs) with a size of 20-100 nm are released by different types of cells in culture media. In addition to proteins, exosomes are enriched with an array of cytokines, specific lipid rafts such as phosphoglyceride, cholesterol, ceramide, fatty-acyl chains, as well as mRNAs, miRNAs, non-coding RNAs, tRNAs, rRNAs, and rarely DNA.
  The purpose of this experimental research is to investigate the effect of exosomes derived from the bone marrow stem cells of small laboratory mice on the maturation of preantral follicles. Material and methods: Exosomes were isolated and cultured from the mesenchymal stem cells of the bone marrow of small laboratory mice by the flushing method. Identification of stem cells was done by flow cytometry method and separation and purification of exosomes was done by ultracentrifuge, identification of exosome was also checked by atomic force microscope (AFM). The effects of exosomes on the viability of follicles were measured by the MTT method, and developmental parameters such as the diameter and the formation of the antrum cavity in the follicles were examined and the follicles were examined on days 0, 2, 3 and 4 of culture with 20 magnification and inverted microscope. Photographs were taken and the diameter of the follicles was measured in micrometers by Image J software. The expression of GDF-9, BMP-15 and BMP-7 genes as genes involved in the growth and maturation of follicles was investigated using Real Time-PCR method. GraphPad Prism 8 software and one way Anova statistical test were used to analyze the data of this research.
Results: The evaluation of the viability of the follicles showed that compared to the control group, the follicles treated with exosome 25, 50 and 100 micrograms/ml showed an increase in viability, as well as the rate of antrum formation increased significantly in the group with the concentration 100 μg/ml showed a significant level (p<0.01**) compared to the control group. The diameter of the follicles increased with increasing the concentration of exosomes compared to the control group. GDF-9, BPM-15 and BMP-7 genes also increased in the treatment groups. Conclusion: According to the findings of this experimental research, it can be stated that exosomes derived from bone marrow stem cells Small laboratory mice have a positive effect on survival and maturity, as well as the growth of ovarian follicles.

Abstract Aim: The stinging nettle (Urtica dioica L.) is a very important medicinal plant that is used in the treatment of many diseases. Considering the important medicinal properties (flavonoid compounds and antioxidant enzymes) of nettle and the numerous advantages of using elicitors in plant tissue culture to enhance the production of medicinal compounds, this study investigated the effect of different concentrations of the chitosan elicitor on the production of some secondary metabolites and antioxidant enzyme activities in callus suspension culture of nettle.
Material and methods: Nettle seeds were cultured in 1/2MS medium after surface sterilization (with 1% sodium hypochlorite and 70% ethanol). leaf explants were isolated from sterile seedling (at the four-leaf stage). The explants were cultured in MS hormone-containing medium (NAA and BA 2.5 and 1 mg L^-1, respectively) and maintained under light conditions of 16 : 8 (light : darkness) hours  at 25 °C. After several stages of subculturing, uniform callus was produced. Eight days after transfer of calli to MS liquid culture medium, calli were treated with chitosan elicitor at concentrations of 0, 50, and 100 mg L^-1. Calli were sampled at 24, 48, and 120 hours after treatment. Finally, the production of secondary metabolites (quercetin, kaempferol, and rutin) was determined using HPLC and the standard line equation. Also, the activities of antioxidant enzymes (phenylalanine ammonia-lyase, polyphenol oxidase, and peroxidase) and the total content of phenol, flavonoid and protein were measured and analyzed by spectrophotometry method. The experiment was conducted in completely randomized design with three replications and was analyzed as a split-time statistical design.
Results: According to the results of the analysis of variance, the effect of chitosan elicitor concentration, sampling time after elicitor application and the interaction effect of elicitor concentration and sampling time on all measured parameters except for the total flavonoid content (the effect of elicitor concentration was not significant) were significant. The highest phenylalanine ammonia-lyase and polyphenol oxidase enzyme activities were at sampling times of 120 and 24 hours after elicitor application at 100 mg L^-1 chitosan concentration, respectively. The peroxidase enzyme activity at the sampling time of 120 hours and a concentration of 50 mg L^-1 of chitosan was 2.57 times that of the control sample. The total protein content decreased at concentrations of 50 and 100 mg L^-1 chitosan compared with the control. The highest amount of quercetin production was in the treatment of 100 mg L^-1 chitosan and sampling time of 48 hours after elicitor application, which increased 25 times compared with the control. Kaempferol and rutin had the highest production at sampling times of 48 and 24 hours and a concentration of 50 mg L^-1, respectively, which increased by 72.6 and 51.2 percent compared with the control.
Conclusion: Chitosan elicitor is known as a biotic elicitor with a positive effect on medicinal and antioxidant properties in various plants. In this study, chitosan elicitor increased the production of quercetin, kaempferol, and rutin metabolites as well as the activity of antioxidant enzymes in nettle callus culture. So, chitosan elicitor can be introduced as an enhancer of medicinal properties of nettle plant under in vitro culture conditions. Obviously, for the commercial production of medicinal compounds, the conditions for large-scale callus production in bioreactors must be optimized.

Abstract Introduction: Knowledge of spatial genetic structures provides a valuable tool for inferring the evolutionary forces such as selective pressures and drift. Low gene flow due to spatial isolation of populations may even increase the degree of local differentiation. Nevertheless, phenotypic plasticity rather than genetic differentiation may be an alternative way of matching genotypes to environment; indeed increasing environmental variation favors higher levels of plasticity.Genetic diversity is one aspect of biological diversity that is extremely important for conservation strategies, especially in rare and narrowly endemic species. The genus Hesperis L. (Brassicaceae) comprises biennial and perennial herbs and consists of 46 species worldwide, mainly occurring in different parts of Europe, Caucasus, Transcaucasia, and to a lesser extent in northern and central Asia, and mostly in Turkey with 28 species. The genus is represented by 11 or six species belonging to sections Hesperis Dvořák, Diaplictos Dvořák and Pachycarpos Fourn. in Iran.
Aim: Moreover, due to extensive morphological variability of this species in the country, there is possibility of having infra-specific taxonomic forms in this species. Therefore, we carried out population genetic analysis and morphometric study of 11 geographical populations for the first time in the country. For genetic study, we used the inter-retrotransposon amplified polymorphism (IRAP) method that displays insertional polymorphisms by amplifying the segments of DNA between two retrotransposons. It has been used in numerous studies of genetic diversity. The objectives of this research were to study genetic diversity Hesperis persica with a different geographical origin by inter-retrotransposon amplified polymorphism (IRAP) method.
Materials and Methods: A total of 73 individuals were sampled representing 11 natural populations of H. persica Boiss. subsp. persica and H. persica subsp. kurdica (F. Dvořák & Hadac) F. Dvořák, in Mazandaran, East Azerbaijan, Kohgilouye-Boirahmad, Chaharmahal Bakhtiari, Fars, Zanjan, Tehran, Kordestan, Provinces of Iran during July-Agust 2019-2024.
Fresh leaves were used randomly from 6-10 plants in each of the studied populations. These were dried by silica gel powder. CTAB activated charcoal protocol was used to extract genomic DNA. The quality of extracted DNA was examined by running on 0.8% agarose gel. A set of six outward-facing LTR primers were used for IRAP analysis. We also used 15 different combinations of outward-facing LTR pair primers. PCR reactions were carried in a 25μl volume containing 10 mM Tris-HCl buffer at pH 8; 50 mM KCl; 1.5 mM MgCl2; 0.2 mM of each dNTP (Bioron, Germany); 0.2 μM of a single primer; 20 ng genomic DNA and 3 U of Taq DNA polymerase (Bioron, Germany). The thermal program was carried out with an initial denaturation for 1 min at 94°C, followed by 40 cycles in three segments: 35 s at 95°C, 40s at 47°C and 55s at 72°C. Final extension was performed at 72°C for 5 min. The amplification products were observed by running on 1% agarose gel, followed by the ethidium bromide staining. The fragment size was estimated by using a 100 bp molecular size ladder (Fermentas, Germany).The IRAP profiles obtained for each samples were scored as binary characters. Parameter like Nei’s gene diversity (H), Shannon information index (I), number of effective alleles, and percentage of polymorphism were determined.Nei’s genetic distance among populations was used for Neighbor Joining (NJ) clustering and Neighbor-Net networking. Mantel test checked the correlation between geographical and genetic distance of the studied populations . These analyses were done by PAST ver. 2.17 , DARwin ver. 5 (2012) and SplitsTree4 V4.13.1 (2013) software. AMOVA (Analysis of molecular variance) test (with 1000 permutations) as implemented in GenAlex 6.4, and Nei^,s Gst analysis as implemented in GenoDive ver.2 (2013) were used to show genetic difference of the populations. Moreover, populations^, genetic differentiation was studied by G'ST est = standardized measure of genetic differentiation, and D_est = Jost measure of differentiation. The genetic structure of populations was studied by Bayesian based model STRUCTURE analysis , and maximum likelihood-based method of K-Means clustering of GenoDive ver. 2. (2013). For STRUCTURE analysis, data were scored as dominant markers. The Evanno test was performed on STRUCTURE result to determine proper number of K by using delta K value. In K-Means clustering, two summary statistics, pseudo-F, and Bayesian Information Criterion (BIC), provide the best fit for k.

Results: The highest value of percentage polymorphism (57.41%) was observed in Mazandaran, 30 km S. of Ramsar between Kash-e Chal mountain and Miankuh (population No.8, H. persica subsp. kurdica) which shows high value for gene diversity (0.34) and Shanon, information index (0.43). Population Chaharmahal Bakhtiari, Shahr-e Kurd, tang-e Sayyad protected area, Pir kuh mountain (No.3, H. persica subsp. persica) has the lowest value for percentage of polymorphism (28.11%) and the lowest value for Shanon, information index (0.088), and He (0.022).AMOVA (PhiPT = 0.98, P = 0.010), and Gst analysis (0.654, p = 0.001) revealed significant difference among the studied populations. It also revealed that, 40% of total genetic variability was due to within population diversity and 60% was due to among population genetic differentiation. Pairwise AMOVA produced significant difference among the studied populations. Moreover, we got high values for Hedrick standardized fixation index after 999 permutation (G’st = 0.654, P = 0.001) and Jost, differentiation index (D-est = 0.769, P = 0.001). These results indicate that the geographical populations of Hesperis persica are genetically differentiated from each other. The mean Nm = 0.455 was obtained for all IRAP loci, which indicates low amount of gene flow among the populations. Population assignment test also agreed with Nm result and could not identify significant gene flow among these populations. However, reticulogram obtained based on the least square method, revealed some amount of shared alleles among populations 2 and 3, and between 7 and 8, also between 1, and 4. This result is in agreement with grouping we obtained with PCoA plot, as these populations were placed close to each other. As evidenced by STRUCTURE plot based on admixture model, these shared alleles comprise very limited part of the genomes in these populations and all these results are in agreement in showing high degree of genetic stratification within Hesperis persica populations. In total 76 IRAP bands (loci) were obtained, out of which 14 bands were private. Populations 2 and 5-7 contained 2-5 private bands.
Conclusion: PCoA plot of populations was in agreement with WARD clustering of molecular data. These results indicated that geographical populations of Hesperis persica are well differentiated based on (IRAP) markers.

Abstract Aim: For many years, medicinal plants have been used to treat many diseases, including diabetes and its side effects, due to their natural and antioxidant compounds. One of these systems that suffers from the complications of diabetes is the reproductive system. Many researches have been conducted to investigate the effectiveness of different medicinal plants on the reproductive system. In this study, the effect of Ferula assa-foetida L. gum extract on the spermatogenesis indices and testicular tissue structure of diabetic male Wistar rats was investigated.
Material and Methods: In our study, 42 male Wistar rats were divided into 6 groups. Diabetes induced by a single dose of streptozotocin (55 mg/kg body weight). Evaluation of the effect of Ferula assa-foetida L. oleo gum extract was done by treating animals with gum extract (150 and 250 mg/kg b. w, gavage) for 42 days. After this period, the animals were anesthetized and sacrificed. After this period, the animals were anesthetized and sacrificed. Then the testicular tissue sampling was done and the samples were fixed in 10% formalin, then the preparation steps of the microscopic sections were performed included 1-dehydration, 2-clarification, 3-smearing, 5-paraffin molding, 6- Cutting with a microtome machine, 7- sticking the samples on a slide, 8- staining with hematoxylin and eosin, 9- mounting. Finally, the samples were prepared for microscopic study. The number of germinal epithelium cells of spermatogenic tubules was counted and Spermiogenesis Index (SI), Tubular Differentiation Index (TDI), Meiosis Index (MI) and Sertoli Cell Index (SCI) were measured.
Results: Results showed that Ferula assa-foetida L. oleo gum extract improves the tissue damages of diabetes on testicular tissue and spermatogenesis indices.
Conclusion: Ferula assa-foetida L. oleo gum extract reduces testicular tissue damage caused by diabetes and has an improving effect on spermatogenesis and fertility disorders related to diabetes.